A NOVEL IMMUNOASSAY FOR THE QUANTIFICATION OF HUMAN TISSUE FACTOR-BINDING TO ACTIVATED FACTOR-VII

The binding of tissue factor to factors VII and VIIa (VII/VIIa) is the primary step for coagulation activation. Variants of human tissue fac tor leading to alterations in the binding to factor VII/VIIa have not been reported. We hypothesize that increased or decreased binding of t issue factor to factor VIIa might result in thrombosis and bleeding, r espectively. The aim of this study was to establish an enzyme-linked i mmunosorbent assay to detect abnormalities in the binding of human tis sue factor to recombinant factor VIIa (rVIIa). Tissue factor obtained from human monocytes was bound to rVIIa on microtiter wells in the pre sence of calcium. A murine antibody against human tissue factor and a biotinylated goat anti-mouse immunoglobuline were added. After incubat ion with streptavidin-horseradish peroxidase, colour development was m easured using a chromogenic indicator system. Optimal assay conditions were obtained at tissue factor concentrations of 50-1500 pg/ml and rV IIa concentrations of 2.5 mu g/ml. The binding of tissue factor to rVI Ia was calcium-dependent and was inhibited by a monoclonal tissue fact or antibody directed against the binding sites of tissue factor to rVI Ia. The assay was evaluated in 23 healthy volunteers. Intra-and intera ssay variabilities were 3.9% and 10.2%, respectively. Among 22 subject s with unexplained bleeding and 47 patients with unexplained thrombosi s, an individual with a decreased or increased binding of tissue facto r to rVIIa could not yet be identified. In conclusion, this novel enzy me-linked immunosorbent assay can be used to detect and quantify an in creased or a decreased binding of human tissue factor to rVIIa. Studie s in patients indicate that, if such a defect exists, it is not a comm on cause of thrombosis or bleeding. (C) 1998 Lippincott-Raven Publishe rs.